Rna amplification method, rna detection method and assay kit

ABSTRACT

In general, according to one embodiment, a method containing, reverse-transcribing the first sequence in the target RNA, to obtain a reverse-transcription product containing a first&#39; sequence complementary to the first sequence, dissociating the reverse-transcription product from the target RNA, hybridizing an elongation primer and the reverse-transcription product to elongate both, thereby obtain an elongation product, and maintaining the amplification reaction liquid containing the elongation product, a primer set and Tin(exo-) DMA polymerase and/or Bsm DMA polymerase under an amplification reaction condition, to amplify the first&#39; sequence.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/508,044, filed on Feb. 28, 2018, which is based upon and claims thebenefit of priority from Japanese Patent Application No. 2017-054577,filed Mar. 21, 2017, the entire contents of which are incorporatedherein by reference.

FIELD

Embodiments described herein relate generally to an RNA amplificationmethod, an RNA detection method, and an assay kit.

BACKGROUND

MicroRNA (miRNA) a kind of RNA which does not. code a protein, butserves to for example, control expression of genes or proteins. Thelatest research shows that there is a correlation between the amount ofexpression of miRNA and each of various diseases including cancer.Therefore, miRNA is expected as new biomarker to diseases. Generally,the detection of miRNA is carried out by the Northern blotting,microarray or real time PCR. But, since miRNA is a short single-strandedRNA of about 20 bases, it is difficult to amplify or detect it, or toimprove the sensitivity of the detection. Under such circumstances,there is a demand for development of an miRNA detection method withhigher accuracy.

BRIEF DESCRIPTION OF TEE DRAWINGS

FIG. 1 is a flowchart illustrating an example of an RNA amplificationmethod of an embodiment.

FIG. 2 is a conceptual diagram showing an example of a target RNA and areverse-transcription primer.

FIG. 3 is a conceptual diagram showing an example of a process ofproducing a reverse-transcription product.

FIG. 4 is a conceptual diagram showing an example of areverse-transcription product and an elongation primer.

FIG. 5 is a conceptual diagram showing an example of an elongationproduct.

FIG. 6 is a conceptual diagram showing an example of a process ofproducing an elongation product.

FIG. 7 is a schematic diagram showing elongation products and primerswhich respectively correspond to each other.

FIG. 8 is a flowchart illustrating an example of an RNA detection methodof an embodiment.

FIG. 9 includes graphs and electropherograms showing test results ofExample 1.

FIG. 10 includes graphs and electropherograms showing test results ofExample 2.

DETAILED DESCRIPTION

In general, according to one embodiment, a method of amplifying a targetRNA containing a first sequence, in a sample, comprises the followingsteps. In step (a), the first sequence in the target RNA isreverse-transcribed, thereby obtaining a reverse-transcription productcontaining a first' sequence complementary to the first sequence. Instep (b), the reverse-transcription product and the target RNA aredissociated from each other. In step (c), an elongation primer and thereverse-transcription product are hybridized to elongate both, therebyobtaining a double-stranded DNA elongation product containing the first'sequence and/or the complementary sequence. In step (d), theamplification reaction liquid containing the elongation product, aprimer set to be bound to the elongation produce and at least one ofTin(exo-) DNA polymerase and Bsm DNA polymerase are maintained under anamplification reaction condition, thereby amplifying the first' sequenceand/or the complementary sequence.

Various embodiments will be described below with reference to theaccompanying drawings. Each figure is an exemplary diagram of anembodiment to aid understanding of the embodiment. The shapes,dimensions or ratios in the drawings may differ from those of the actualdevice, and may be appropriately changed in light of the subsequentexplanation and the known art.

1. RNA Amplification Method

The RNA amplification method according to the embodiment is a method ofamplifying a target RNA in a sample, which contains a first sequence.

The target RNA is RNA to be detected in the RNA amplification method ofthe embodiment. The target RNA preferably is a single-strandedshort-chain RNA having a base length of about 50 bases or less. Theshort-chain RNA may be, for example, a functional RNA such as microRNA(miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA) orsmall nucleolar RNA (snoRNA), or may not be a functional RNA, or may bean artificial RNA or an RNA produced by fragmenting RNA longer than 50bases.

The target RNA contains a first sequence. The first sequence is asequence which can serve as an indicator for detecting the target RNA.That is, the first sequence is a target of reverse-transcriptionreaction in the RNA amplification method of the embodiment. Note thatthe first' sequence, which is a complementary sequence to the firstsequence, and a complementary sequence thereto, produced byreverse-transcription of the first sequence are the targets of theamplification reaction in the RNA amplification method of theembodiment. For example, the first sequence may be a sequence containedin the target RNA and also specific to the target RNA. The firstsequence may be the sequence ranging over the entire length of thetarget RNA, or a partial continuous sequence selected from the sequenceranging over the entire length of the target RNA. The base length of thefirst sequence can be, for example, 3 to 10 bases, 10 to 20 bases, 20 to30 bases, 30 to 40 bases, or 40 to 50 bases, and preferably 10 to 50bases.

Here, the term “amplification” refers to continuously reproducing atemplate nucleic acid using a primer set and an enzyme. Theamplification method employable in the embodiment can be anyamplification method conventionally well-known by itself which amplifiesnucleic acid using a primer set. The amplification method may be, forexample, an isothermal amplification method. The isothermalamplification method may be, for example, LAMP, SMAP, RCA, ICAN or thelike, although it is not limited to these.

The RNA amplification method of the embodiment will be described withreference to FIG. 1. FIG. 1 briefly shows the flow of the RNAamplification method.

In step (a), the first sequence is reverse-transcribed, and thus areverse-transcription product containing the first' sequencecomplementary to the first sequence is obtained.

Step (a) may be carried out by, for example. maintaining areverse-transcription reaction liquid under a reverse-transcriptionreaction condition. The reverse-transcription reaction liquid includes asample, a reverse-transcription primer and a reverse transcriptase.

The sample is an object to be analyzed and is one which may contain thetarget RNA. The sample may be, for example, a material such as blood,serum, leukocyte, lymph, spinal fluid, urine, feces, semen, sweat,saliva, oral mucosa, expectoration, lacrimal fluid, mother milk,amniotic fluid, tissue, biopsy, isolated cells or cultured cellsextracted from an animal, or an organ, isolated cells or cultured cellsor an extract from a plant, or a mixture containing microbe, bacteria,fungus or virus, or an environmental material extracted fromenvironment, a mixture containing synthetic RNA, or a material itself ofa mixture containing any of these. Or it may be a formulation preparedfrom any of these materials.

The animal may be, for example, a mammal, bird, amphibian, reptile,fish, or arthropod, or some other living organism belonging to theanimal kingdom. The mammal may be any of a primate such as an ape orhuman, a rodent such as a mouse or rat, a companion animal such as adog, cat or rabbit, or a livestock such as a horses, cow or pig.

It is preferable that the sample be in a state that does not interferewith the amplification reaction. Being in such a state, it is possibleto amplify the target RNA more efficiently in the RNA amplificationmethod of the embodiment. The state which does not interfere with theamplification reaction is defined as, for example, that in which thekind of an ingredient in the sample, a combination of ingredients, orthe concentration of an ingredient does not decrease the amplificationreaction speed, or delay or stop the reaction.

When the material itself, described above is in the state which does notinterfere with the amplification reaction, the material may be useddirectly as a sample. Or, for example, the obtained material may besubjected to a pretreatment conventionally well-known by itself toobtain a sample of the state which does not interfere with theamplification reaction or of a more suitable state for theamplification. The pretreatment is, for example, mincing,homogenization, centrifuging, precipitation, extraction and/orseparation.

For example, the extraction may be carried out with use of acommercially available nucleic acid extraction kit. Usable examples ofthe nucleic acid extraction kit are ureLink (registered trademark) miRNAIsolation Kit (of Thermo Fisher Scientific), microRNAExtractor(registered trademark) SP Kit (of Wako Pure ChemicalIndustries, Ltd.), NucleoSpin (registered trademark) miRNA (of TakaraBio Inc.), mirpremier (registered trademark) microRNA Isolation Kit (ofSigma-Aldrich Co.), High Pure miRNA Isolation Kit (of Roche LifeScience, Ltd.) and PAXgene Blood miRNA Kit (of Qiagen, Ltd.), but arenot limited to these. Alternatively, a sample may be obtained withoutusing such a kit, but by, for example, diluting the material with abuffer solution, followed by heat-treatment at 80° C. to 100° C. andextraction by centrifuging the resultant and collecting the supernatant.

The reverse-transcription primer is a primer of a single-stranded DMA toreverse-transcribe the first sequence. FIG. 2 shows an example of thetarget RNA and the reverse-transcription primer of the embodiment. Thetarget RNA (i) contains the first sequence. The reverse-transcriptionprimer (ii) contains a reverse-transcription primer sequence and a firstamplification sequence. The reverse-transcription primer sequence andthe first amplification sequence are contained in thereverse-transcription primer in this order from a 3′ to a 5′ direction.The full length of the reverse-transcription primer should preferably be20 to 80 bases.

The reverse-transcription primer sequence is a sequence which serves asa primer for reverse-transcribing the first sequence. The base sequenceof the reverse-transcription primer sequence is a sequence complementaryto a desired sequence on the target RNA, which is selected so that thefirst sequence of the target RNA (i) can be reverse-transcribed over itsfull length. The reverse-transcription primer sequence is, for example,a sequence complementary to a continuous sequence containing a 3′terminal of the first sequence on the target RNA. Or, when the firstsequence is selected to have a desired number of sequences between a 3′terminal of the target RNA and a 3′ terminal of the first sequence asshown in FIG. 2, part (i), the reverse-transcription primer sequence maybe complementary to the sequence between the 3′ terminal of the firstsequence and the 3′ terminal of the target RNA (not shown). The baselength of the reverse-transcription primer sequence may be 5 to 20bases, though not particularly limited, but preferably, 7 to 15 bases,or more preferably, 3 to 12 bases.

The first amplification sequence is a sequence noncomplementary to thefirst sequence, and contains a sequence or a complementary sequencethereof, which may be bound to at least one primer contained in theprimer set to which bound to the elongation product, which will bedescribed later. Besides such a sequence, the first amplificationsequence may contain a predetermined sequence, which may be, forexample, such a sequence that the sequence itself or a complementarysequence thereof is necessary for the amplification reaction in the RNAamplification method of the embodiment. The base length of the firstamplification sequence should preferably be, for example, 20 to 60bases.

For example, after designing, in advance, the base sequence of theprimer contained in the primer set to be bound to the elongation productin step (c), which will be described later, based on the structure andbase sequence of the primer, the base sequence of the firstamplification sequence may be designed. Or, a DNA sequence of a desiredlength may be selected as the first amplification sequence, and theprimer set to be bound to the elongation product may be designed basedon the base sequence thereof.

A spacer sequence may be present between the reverse-transcriptionprimer sequence and the first, amplification sequence. The base lengthof the spacer sequence should preferably be, for example, 4 to 16 bases.

The reverse transcriptase may be any of the well-known types, andselected according to, for example, the type of thereverse-transcription primer and/or the type or sequence of the targetRNA. Examples of the reverse transcriptase are M-MuLV reversetranscriptase, AMV reverse transcriptase, transcriptor reversetranscriptase, Superscript (registered trademark) transcriptor reversetranscriptase, or MultiScribe reverse transcriptase, though not limitedto these.

The reverse-transcription reaction liquid may contain, in addition tothese ingredients, ingredients necessary for the reverse-transcriptionreaction. Such ingredients may be, for example, a substrate such as asalt or deoxynucieoside triphosphates (dNTPs), a thickening agent as areaction reagent, a buffer material for adjusting pH, a surfactant, anion which increases the annealing specificity and or an ion which givesrise to a cofactor of reverse transcriptase, etc.

The reverse-transcription reaction liquid described above is maintainedunder reverse-transcription reaction conditions. Thereverse-transcription reaction conditions may be selected depending onthe type of the reverse-transcription primer, the type of the target RNAand/or the type of the reverse transcriptase, etc., based on the commonknowledge of a person having ordinary skilled in the art. Examples ofthe conditions for the reverse-transcription reaction may be atemperature of 45° C. or less and for 15 minutes to 1 hour.

When the reverse-transcription reaction liquid is maintained under theseconditions, the first sequence is reverse-transcribed, and areverse-transcription product containing the first' sequencecomplementary to the first sequence is obtained.

FIG. 3 shows an example of the process of producing areverse-transcription product. The example shown in FIG. 3 is the casewhere the LAMP method is used in the RNA amplification method of theembodiment.

First, a reverse-transcription primer sequence 4 of areverse-transcription primer 3 hybridizes with a first sequence 2 of atarget RNA 1. The reverse-transcription primer 3 contains thereverse-transcription primer sequence 4, and a B1 sequence, a B2sequence and a B3 sequence as the first amplification sequence, in thisorder. The B1 sequence, B2 sequence, and B3 sequence are designed basedon, for example, the LAMP primer set used in a later step of theisothermal amplification. The LAMP primer set includes a BIP primercontaining a B2 sequence and a B1c sequence, an FTP primer containing anF2 sequence and an F1c sequence, a B3 primer containing a B3 sequenceand an F3 primer containing an F3 sequence (not shown). Here, the terms“B1 sequence”, “32 sequence”, “B3 sequence”, “F1 sequence”, “F2sequence” and “F3 sequence” are used in the same meanings as those usedby a person skilled in the art when designing a LAMP primer. Further,the B1c sequence is complementary to the B1 sequence, and the F1csequence is complementary to the F1 sequence. The B2 sequence in thefirst amplification sequence is a complementary sequence of a sequenceto which the B2 sequence of the BIP primer binds. The B3 sequence in thefirst amplification sequence is a complementary sequence of a sequenceto which the B3 sequence of the B3 primer binds. The B1 sequence in thefirst amplification sequence is a sequence necessary for producing anamplification product in the LAMP reaction.

Next, with a reverse transcriptase (not shown), the elongation to the 3′terminal of the reverse-transcription primer sequence 4 in a 5′direction (indicated by hollow arrow), which uses the target RNA 1 asthe template, advances. Thus, the first sequence 2 isreverse-transcribed to produce a first' sequence 5 complementary to thefirst sequence 2. As a result, a reverse-transcription product 6 whichincludes the first' sequence 5, the B1 sequence, B2 sequence and B3sequence in this order is obtained. The reverse-transcription product 6is hybridized with the target RNA 1.

Next, in a step (b), the reverse-transcription product and the targetRNA are dissociated from each other. The dissociation may be carriedout, for example, after the reverse-transcription reaction by heatingthe reverse-transcription reaction liquid to 80° C. to 100° C. With suchheating, the reverse transcriptase may be deactivated at the same timeas the dissociation of the RNA described above. Thus, it is possible toprevent the reverse-transcription reaction from adversely affecting thelater steps after step (b).

Next, in step (c), the elongation primer and the reverse-transcriptionproduct are hybridized to elongate the elongation primer and thereverse-transcription product, thereby obtaining an elongation productof double-stranded DMA which contains the first' sequence and/or itscomplementary sequence.

Step (c) will be described in more detail with reference to FIGS. 4, 5and 6.

Step (c) may be carried out, for example, by maintaining the elongationreaction liquid under elongation reaction conditions. The elongationreaction liquid contains a reverse-transcription product obtained insteps (a) and (b), an elongation primer and a DNA polymerase.

The reverse-transcription product is a reverse-transcription productobtained in steps (a) and (b).

The elongation primer is a single-stranded DNA and is a primer whichhybridizes with a reverse-transcription product for obtaining adouble-stranded DNA which can be amplified in step (d). FIG. 4 shows anexample of each of the reverse-transcription product and the elongationprimer. The reverse-transcription product (iii) contains the first1sequence and the first amplification sequence. The elongation primer(iv) contains an elongation primer sequence and a second amplificationsequence. The elongation primer sequence and the second amplificationsequence are contained in the elongation primer in this order from the3′ toward 5′ direction.

The elongation primer sequence is a sequence selected so as to be ableto produce a complementary sequence of a sequence ranging over theentire length of the first' sequence using the first' sequence as thetemplate, and it is a sequence complementary to a continuous sequencecontaining the 3′ terminal of the reverse-transcription product (iii).The elongation primer sequence may also include a sequence complementaryto a sequence of 5 bases or less, preferably, 3 bases or less, from the3′ terminal of the reverse-transcription primer to the 5′ direction. Thebase length of the elongation primer sequence should preferably be, forexample, 30 to 120 bases.

The second amplification sequence contains a sequence which isnoncomplementary to the first' sequence, and also is to which a primerto be bound to the elongation product can bind, or complementarysequence thereof. The second amplification sequence may contain apredetermined sequence in addition to the sequence to which a primercontained in the primer set to be bound to the elongation product, whichwill be described later, can bind or a complementary sequence thereof.It may be, for example, a sequence, itself or a complementary sequencethereof, necessary for the amplification reaction. The base length ofthe second amplification sequence should preferably be, for example, 30to 90 bases.

For example, after designing the base sequence of the primer set to bebound to the elongation product used at a step (c), based on thestructure of the primer set, the base sequence of the secondamplification sequence may be designed. Or, a DNA sequence of a desiredlength may be selected as the second amplification sequence, and theprimer to be bound to the elongation product may be designed based onthe base sequence thereof.

A spacer sequence may be present between each sequence pair of anelongation primer sequence and the second amplification sequence. Thebase length of the spacer sequence should preferably be, for example, 4to 16 bases.

The DNA polymerase may be any well-known reverse DNA polymerase, and isselected according to the type of the elongation primer and/or thesequence of the reverse-transcription product, etc. Usable examples ofsuch DNA polymerase are Klenow Fragment (Large Fragment E. coli DNApolymerase I), T4 DNA polymerase, phi29 DNA polymerase, Bst. DMApolymerase, Csa DNA polymerase, 96-7 DNA Polymerase, Vent(exo-) DNApolymerase, Gsp SSD DNA polymerase, Tin exo-DNA polymerase, and alsoother DNA polymerases generally used for the PCR amplification includingTaq DNA polymerase. Or, reverse transcriptases which can carry out theamplification using DNA as the template, such as M-MuLV ReverseTranscriptase, Transcriptor Reverse Transcriptase or the like can beused. Further, DNA polymerase may be at least one of Tin(exo-) DNApolymerase and Bsm DNA polymerase used in step (d), as will be describedin detail.

In addition to these ingredients, the elongation reaction liquid mayfurther contain a predetermined ingredient necessary for the elongationreaction. Such ingredients may be, for example, a substrate such as saltand dNTPs, a thickening agent as a reaction reagent, a buffer foradjusting pH, a surfactant, an ion that enhancing the annealingspecificity, and/or an ion used as a cofactor of the reversetranscriptase.

The elongation reaction liquid described above is maintained under anelongation reaction condition. The elongation reaction condition may beselected depending on the type of the elongation primer, the type of thereverse-transcription product, and/or the type of the DNA polymerase,etc., based on the common knowledge of a person having an ordinary skillin the art. The reaction temperature of the elongation reaction isdependent on the type of enzyme, and is, generally, for example, 10° C.to 80° C. The elongation reaction can be carried out by maintaining thereaction temperature at constant, in a plurality of temperature zoneseach for a certain period of time, or repeating a plurality oftemperature zones in a plurality of times of cycles.

By maintaining the elongation reaction liquid under these conditions,the elongation primer hybridizes to the reverse-transcription product,and the elongation primer and the reverse-transcription product elongatewhile utilizing each other as a template, and thus the elongationproduct of a double-stranded DNA can be obtained. FIG. 5 shows anexample of the elongation product. One chain of the elongation product(v) contains a complementary sequence of the second amplificationsequence, the first' sequence and the first amplification sequence, andthe other chain contains the second amplification sequence, acomplementary sequence of the first' sequence and a complementarysequence of the first amplification sequence.

FIG. 6 shows an example of the process of obtaining an elongationproduct from the reverse-transcription product 6 shown in FIG. 3, whichhas dissociated from the target RNA 1.

First, to a first' sequence 5 of the reverse-transcription product 6, anelongation primer sequence 9 of an elongation primer 8 hybridizes. Theelongation primer 8 contains the elongation primer sequence 9 and F1sequence, F2 sequence and F3 sequence as the second amplificationsequence.

The F1 sequence, F2 sequence and F3 sequence are designed based on theLAMP primer set used in a later step, for example. The LAMP primer setcontains the primers (not shown) described with reference to FIG. 3. TheF2 sequence in the second amplification sequence is a complementarysequence of a sequence to which the F2 sequence of the FIP primer binds.The F3 sequence in the second amplification sequence is a complementarysequence of a sequence to which the F3 sequence of the F3 primer binds.The F1 sequence in the second amplification sequence is a sequencenecessary to produce the amplification product in the LAMP reaction.

Next, with the DNA polymerase, the elongation primer 8 and thereverse-transcription product 6 elongate while utilizing each other as atemplate. That is, the 3′ terminal of the elongation primer 8 elongatesusing the reverse-transcription product 6 as the template, and at thesame time, the 5′ terminal of the reverse-transcription product 6elongates using the elongation primer 8 as the template (as indicated bya hollow arrow). As a result, an elongation product 10 ofdouble-stranded DNA is produced. The elongation product 10 contains, inone chain, F1c to F3c sequences complementary to the F1 to F3 sequences,respectively, the first' sequence 5 and B1 to B3 sequences, and in theother chain, F1 to F3 sequences, a sequence 11 complementary to thefirst' sequence 5 and B1c to B3c sequences complementary to the B1 to B3sequences, respectively.

In step (d), the amplification reaction liquid containing the elongationproduct obtained in step (c), the primer set to be bound to theelongation product and at least one of Tin(exo-) DNA polymerase and BsmDNA polymerase is maintained under the amplification reactionconditions, thereby amplifying the first′ sequence and/or itscomplementary sequence by using the elongation product as the template.

The ingredients for the amplification reaction contained in theamplification reaction liquid will be described.

The elongation product is one obtained in step (c).

The primer set to be bound to the elongation product is a set of primersnecessary for amplifying the first' sequence or complementary sequencethereof by using the elongation product as a template. The primer set tobe bound to an elongation product may be, for example, an isothermalamplification primer set. The isothermal amplification primer setcontains at least the first primer for binding to the firstamplification sequence or its complementary sequence and the secondprimer for binding to the second amplification sequence or itscomplementary sequence.

The primer set to be bound to the elongation product may be designed,when the base sequences of the reverse-transcription primer and theelongation primer are predetermined, based on the base sequences of thereverse-transcription primer and the elongation primer and the type ofthe amplification method employed according to the common knowledge of aperson skilled in the art. When designing the base sequence of theprimer set to be bound to an elongation product before determining thebase sequences of the reverse-transcription primer and the elongationprimer, the primer set to be bound to the elongation product, may bedesigned based on the type of the amplification method employedaccording to the common knowledge of a person skilled in the art, andthe base sequences of the reverse-transcription primer and theelongation primer may be determined based thereon.

FIG. 7 shows an example of the primer set to be bound to the elongationproduct. The primer set be bound to the elongation product, shown inFIG. 7 is for amplifying the first' sequence 5 and its complementarysequence 5′ of the elongation product 10 shown in FIG. 6. In thisexample, the primer set to be bound to an elongation product is anisothermal amplification primer set, and contains a BIP primer 11, FIPprimer 12, B3 primer 13 and F3 primer 14. The BIP primer 11 contains aB2 sequence and a B1c sequence, and the B2 sequence and B1c sequence arelinked in this order from the 3′ toward the 5′ direction. The B2sequence is a sequence for binding to the B2c sequence contained in thecomplementary sequence of the first amplification sequence. The B1csequence is a sequence complementary to the B1 sequence. The FIP primer12 contains an F2 sequence and an F1c sequence, and the F2 sequence andF1c sequence are linked in this order from the 3′ toward the 5′direction. The F2 sequence is a sequence for binding to the F2c sequencecontained in the complementary sequence of the second amplificationsequence. The F1c sequence is a sequence complementary to the F1sequence. The B3 primer 13 contains a B3 sequence. The F3 primer 14contains an F3 sequence. Even without the F3 region or B3 region, thereaction occurs. The B2 sequence of the BIP primer is a sequence forbinding to the complementary sequence of the B2 sequence in the firstamplification sequence. The F2 sequence of the FIP primer is a sequencefor binding to the complementary sequence of the F2 sequence in thesecond amplification sequence. Therefore, the primer set to be bound toan elongation product, shown in FIG. 7 contains a BIP primer as thefirst primer and an FIP primer as the second primer.

Tin(exo-) DNA polymerase and Bsm DNA, polymerase are each an enzyme forcatalyzing the amplification reaction which amplifies the first'sequence and its complementary sequence using the elongation product asthe template. Tin(exo-) DNA polymerase and Bsm DNA polymerase eachshould only be a conventionally known Strand displacement enzymegenerally called by these names. The amplification reaction liquidcontains Tin(exo-) DNA polymerase, Bsm DNA polymerases, or a combinationof these. The concentration of these enzyme in the amplificationreaction liquid may be 2 U to 32 U.

With use of Tin(exo-) DNA polymerase, Bsm DNA polymerases or acombination of these as the enzyme catalyzing the amplificationreaction, it is possible to more specifically amplify the full length ofthe elongation product containing the first' sequence and thecomplementary sequence. That is, for example, in thereverse-transcription reaction of step (a) and the elongation reactionof step (c), a nonspecific product which do not contain the first'sequence and its complementary sequence or contain only a portionthereof, if any, can be produced in addition to the targetreverse-transcription product and elongation product. If such anonspecific product is amplified, an amplification product that containsa low concentration of the original target amplification productcontaining the full length of the first' sequence and the complementarysequence may be undesirably generated. In this case, it is difficult todetect the target RNA with sufficient accuracy. However, with the methodof the embodiment, with use of Tin(exo-) DNA polymerase and/or Bsm DNApolymerase it is possible to suppress the amplification of thenonspecific product and obtain an amplification product which containssequences of the original target object of the amplification more. Thus,with use of these enzymes, it becomes possible to specifically amplifyan RNA whose content in the sample is very low, which is difficult withthe conventional technique. Thus, an RNA amplification method withsimpler procedure, higher sensitivity and specificity can be provided bya. Further, with use of these, enzymes, the target RNA can bespecifically amplified even under such a condition that a great numberof nonspecific products are present. Therefore, steps (a) to (d) can becarried out in the reaction liquid which serves as all of thereverse-transcription reaction liquid, elongation reaction liquid andamplification reaction liquid, without removing the nonspecific productsgenerated by the reverse-transcription reaction and elongation reaction,or separating the reverse-transcription products for the elongationreaction, or separating the elongation products for the amplificationreaction, as will be described in detail later. Thus, a further simplerRNA amplification method can be provided.

The amplification reaction liquid may contain, in addition to theseingredients, a desired ingredient necessary for the amplificationreaction. Such an ingredient may be, for example, a substrate such asdeozynucleoside triphosphates (dNTPs) or a salt for maintaining anappropriate environment for the amplification or the like.

The condition for the amplification reaction may be selected dependingon the type of the primer or the like based on the common knowledge of aperson skilled in the art. The condition for the amplification reactionmay be, for example, an isothermal amplification condition, which maybe, for example, an isothermal temperature of 50° C. to 70+ C. for 15 to90 minutes.

By maintaining the amplification reaction liquid under the amplificationreaction condition, the first' sequence and/or its complementarysequence are amplified by using the elongation product as the template.

With the RNA amplification method of the embodiment, the target RNA in asample can be amplified more simply, with higher sensitivity andspecificity.

In a further embodiment, the DMA polymerase contained in the elongationreaction liquid of step (c) may contain at least one of Tin(exo-) DNApolymerase and Bsm DNA polymerase, or a combination of these. In thatcase, the DNA polymerase used in step (c) may be used for theamplification reaction in step (d). That is, for example, Tin(exo-) DNApolymerase, Bsm DNA polymerase or a combination thereof may be containedas DNA polymerase in the elongation reaction liquid and step (d) may becarried out after finishing the elongation reaction without adding theenzyme for the amplification to the amplification reaction liquid. Inthis manner, the amount of the enzyme used can be decreased, therebyreducing the cost. Further, the procedure of the test can be morefacilitated.

The reverse-transcription reaction, elongation reaction andamplification reaction, described above can be carried out in thereaction liquid which serves as all of the reverse-transcriptionreaction liquid, elongation reaction liquid and amplification reactionliquid. That is, for example, an ingredient necessary for the elongationreaction and amplification reaction may be added in advance to thereverse-transcription reaction liquid before carrying out thereverse-transcription reaction, or after each reaction, an ingredientnecessary for the next reaction may be added to the reaction liquid, soas to be used in the next reaction. Or, after each reaction, all or partof the reaction liquid may be added to the solution containing aningredient necessary for the next reaction. In that case, it is notnecessary to remove the nonspecific product produced by thereverse-transcription reaction and elongation reaction, separate thereverse-transcription product for the elongation reaction, or separatethe elongation product for the amplification reaction or the like, andthus the operation is simpler. This can be achieved by using Tin(exo-)DMA polymerase or Bsm DMA polymerase which can amplify the targetproduct specifically even under the condition where a nonspecificproduct exists, in the amplification reaction.

2. RNA Detection Method

According to a further embodiment, a target RNA detection method isprovided.

FIG. 8 shows a flow schematically showing an example of the RNAdetection method of the embodiment. The RNA detection method of theembodiment comprising executing steps (a) to (d) of the RNAamplification method of the embodiment, and detecting (step (e)) anamplification product obtained during or after maintaining under theamplification reaction condition in step (d).

The detection of an amplification product can be carried out based on,for example, turbidity, fluorescence, electrochemical signal or the likeas an indicator. The detection of the amplified product using turbidityas an indicator may be performed, for example, by a turbidimeter, anabsorption spectrometer, visual observation or the like. The detectionof the amplified product using fluorescence as an indicator may beperformed, for example, by detecting the fluorescence generated using areagent that produces fluorescence, such as a fluorescence reagentcontaining calcein or intercalater, depending on the presence of theamplified product or the amplification reaction. The detection of theamplified product using electrochemical signal as an indicator may beperformed, for example, by detecting the signal generated using areagent that produces an electrochemical signal of a redox reaction orthe like, such as an intercalator, depending on the presence of theamplified product or the amplification reaction. The detection of anamplification product may be carried out at a specific time from thestart of the amplification reaction or may be carried out with time. Theterm “with time” may be interpreted as continuously or intermittently,in which the detection is carried out at a plurality of times atpredetermined intervals.

Further, the detection of the amplification reaction and/or theamplification product can be carried out by detecting the hybridizationbetween the amplification product and the probe. The hybridization maybe detected by a probe-immobilized substrate. The probe-immobilizedsubstrate comprises a substrate and a plurality of types of probesimmobilized on the substrate. The probes may each include the samesequence as that of each respective detection region, or itscomplementary sequence. With the above-described configuration, a probeis hybridized with an amplification product containing the correspondingsequence. The presence of the amplification product or the amountthereof is detected by sensing the hybridization. Thus, in the detectionmethod, the presence of a target nucleic acid in a sample or the amountthereof is detected.

The probe-immobilized substrates may be interpreted as synonyms of suchterms generally used, as “DNA chip” and “DNA array” in a narrow sense,for example, and may be used exchangeably among them. Further, forexample, devices such as a cassette for detection and a cartridge, whichare prepared by integrating, for example, a small-sizedprobe-immobilized substrate such as a DNA chip and other structuralmembers necessary to form an amplification reaction unit, a passage,etc., into one unit may be interpreted also as probe-immobilizedsubstrates. In that case, the amplification reaction and/or detectionmay be automatically carried out by a device which can automaticallycontrol the temperature within the DNA chip and/or transferring of thesample and the reaction liquid, etc.

From the results of the detection, the presence of the target RNA in thesample and/or the amount thereof can be determined. These items aredetermined, for example, by measuring the time required until theturbidity or fluorescence exceeds a predetermined threshold as the risetime. When the target RNA is present, a rise of the increase inturbidity or fluorescence is observed at an earlier point. Or, theconcentration of the target RNA can be determined based on the risetime. Or, the concentration of the target RNA can foe determined by, forexample, preparing a calibration curve from measurement results of aplurality of different reference samples whose amounts of target RNApresent are already known and comparing the detection results with thecalibration curve.

In the further embodiment of the RNA detection method, the first1sequence and its complementary sequence may be separated from ethersequences by fragmenting the amplification product with a specificrestriction enzyme, and the first' sequence and its complementarysequence may be detected. In that case, the reverse-transcriptionprimer, elongation primer and primer sets to be bound to elongationproduct, are designed so that the amplification product includes asequence which can be broken by a specific restriction enzyme. After theamplification reaction, the amplification product is treated with acorresponding restriction enzyme, and then subjected to, for example,electrophoresis. When it is found that the target RNA is present in thesample and the sequence containing the first' sequence and itscomplementary sequence is present in an amplification product, by theelectrophoresis, the product appears as one band in a specific position.Thus, the presence/absence of a target RNA in a sample can be judgedmore clearly.

According to the RNA detection method of the embodiment, a target RNAcan be detected simply, with high sensitivity and specifically.

Further, when the target RNA is, for example, an RNA to be expressed orwhose amount of expression increases or decreases in a cell afflictedwith a specific disease, by detecting the target RNA using the detectionmethod of the embodiment, it is possible to determine whether or not aliving organism, from which a sample is extracted, is afflicted with aspecific disease more simply, -with higher sensitivity and specificity.Examples of the specific disease may be cancers such as breast cancer,colorectal cancer or lung cancer, or other diseases. Or, for example,when the target RNA is an RNA to be expressed, or whose amount ofexpression increases or decreases in specific bacteria or virus, bydetecting the target RNA using the detection method of the embodiment,it is possible to determine the presence of specific bacteria or virusof a sample or whether or not the living organism, from which a sampleis extracted, is infected by specific bacteria or virus, more simply,with higher sensitivity and specificity.

3. Assay Kit

According to an embodiment, an assay kit for amplifying or detecting atarget RNA containing the first sequence in a sample is provided.

The assay kit of the embodiment comprises a reverse-transcriptionprimer, reverse transcriptase, an elongation primer, a primer set to bebound to an elongation product, and Tin(exo-) DNA polymerase, Bsm DNApolymerase or a combination thereof. The assay kit may contain a furtheringredient necessary for the reverse-transcription reaction, elongationreaction and/or amplification reaction.

The assay kit may further contain a DNA polymerase different fromTin(exo-) DNA polymerase or Bsm DNA polymerase.

The ingredients are those described above. The ingredients may beseparately contained in respective containers or combinations of any ofthese or all the ingredients may be contained in the same container.

According to such an assay kit, it is possible to detect the target RNAmore simply, with higher sensitivity and specificity.

EXAMPLES Example 1 (Comparative Example): Detection of RNA by the LAMPMethod Using Gsp SSD DMA Polymerase, Bst DMA Polymerase and Bst2.0 DMAPolymerase

(1) Reverse-Transcription Reaction

First, a plurality of reverse-transcription reaction liquids (20 μL) ofdifferent concentrations of synthetic miRNA (1E+3, 1E+4, 1E+5, 1E+6, and0 copy) were prepared. The reverse-transcription reaction liquidscontain, in addition to the respective synthetic miRNAs, 5 nM ofreverse-transcription primer, 20 mM of Tris-HCl (pH: 8.8), 50 mM of KCl,8 mM of MgCl₂, 10 mM of (NH₄)₂SO₄, 0.1% of Tween-20, 0.8 M of betaine,1.4 mM of each of dNTPs, 1 mM of DTT and 4 U of RNaseOUT all at thefinal concentration. Each of the reverse-transcription reaction liquidswas maintained at 16° C. for 30 minutes, 42° C. for 30 minutes and 85°C. for 5 minutes, and the reverse-transcription reaction was allowed tooccur.

(2) Elongation Reaction

Then, to each of the reverse-transcription reaction liquids, 4.45 nM ofthe elongation primer and 0.4 U of DeepVent(exo-) were added to prepareelongation reaction liquids. Each of the elongation reaction liquids wasmaintained at 95° C. for 2 minutes, and subjected to 35 cycles of themaintenance (at 95° C. for 20 seconds, 55° C. for 30 seconds and 72° C.for 30 seconds), followed by the maintenance at 72° C. for 5 minutes,thereby allowing the elongation reaction to occur.

(3) LAMP Reaction

To each of the liquids respectively containing, all at the finalconcentration, 20 mM of Tris-HCl (pH: 8.8), 50 mM of KCl, 8 mM of MgCl₂,10 mM of (NH₄)₂SO₄, 0.1% of Tween-20, 0.8 M of betaine, 1.4 mM of eachof dNTPs, 1.6 μM of the FIP primer, 1.6 μM of the SIP primer, 0.8 μM ofthe LF primer, 8 U of Gsp SSD DEA polymerase, the elongation reactionliquid (10 μL) obtained after the elongation reaction was added, toprepare a plurality of 25 μL of LAMP reaction liquids. As a positivecontrol, a reaction liquid containing synthetic DMA (1E+5 copies)containing a sequence of the target elongation product was prepared. Asa negative control, a solution not containing any nucleic acid strandwas prepared. Each of the LAMP reaction liquids, positive control andnegative control were maintained at 65° C. for 90 minutes, to allow theLAMP reaction to occur. For each of the liquids, the turbidity wasmeasured with time.

Similarly, a LAMP reaction liquid containing Bst DNA polymerase orBst2.0 DNA polymerase as the DNA polymerase was prepared and subjectedto the amplification reaction and the detection of turbidity similarly.

(4) Electrophoresis

Subsequently, each of the reaction liquids was treated with therestriction enzyme HinfI to fragment the amplification product, and thensubjected to electrophoresis. The above-described test was carried outwith two tubes (two repetitions) per one type of reaction liquid. Theresults are shown in FIG. 9.

FIG. 9 shows the results with Gsp SSD DNA polymerase in part (a), BstDNA polymerase in part (b) and Bst2.0 DMA polymerase in part (c). Eachpart of the figure indicates the time to threshold of turbidity(hereinafter “Tt”) of each of the reaction liquids described above andthe electropherogram.

The same band pattern as that of the positive control (PC) was obtainedonly with reaction liquids containing 1E+6 copies (10^6) of miRNA whenGsp SSD DNA polymerase and Bst2.0 DMA polymerase were used, and onlywith reaction liquids containing 1E+6 or more (10^5) copies of miRNAwhen Bst DMA polymerase was used. Even with any of these enzymes, whenthe concentration of miRNA is lower than a concentration in which thesame band pattern as that of the positive control (PC) was obtained, theamplification product was produced (the rise in turbidity occurred), butthe sequence of miRNA, the target, was not specifically amplified.

Example 2 (the Embodiment): Detection of Short-Chain RMA by the LAMPMethod Using Tin(exo-) DNA Polymerase or Bsm DNA Polymerase

With a method similar to that of the comparative example, miRNA wasreverse-transcribed, elongated and amplified using Tin(exo-) DNApolymerase or Bsm DMA polymerase as the LAMP enzyme. The results areshown in FIG. 10. FIG. 10 shows the results with Tin(exo-) DNApolymerase in part (a) and those of Bsm DNA polymerase in part (b). Eachpart of the figure indicates the Tt value of each of the reactionliquids described above and the electropherogram.

The same band pattern as that of the positive control (PC) was obtainedwith reaction liquids containing 1E+5 copies (10^5), 1E+4 copies (10^4)and 1E+3 copies (10^3) of miRNA when Tin(exo-) DNA polymerase was used,and with reaction liquids containing 1E+6 copies (10^6), 1E+5 copies(10^5) and 1E+4 copies (10^4) of miRNA when Bsm DNA polymerase was used.Thus, it has been clarified that the target gene can be specificallyamplified from a reaction liquid with less concentration of miRNA in thecase which uses Tin(exo-) DNA polymerase or Bsm DNA polymerase than thecase where Gsp SSD DNA polymerase, Bst DNA polymerase or Bst2.0 DNApolymerase was used.

Example 3: Evaluation in Ampliflability and Specificity of Amplificationof 11 Types of Strand Displacement Enzymes

As to 11 types of Strand displacement enzymes (including those used inExamples 1 and 2) each generally known to have a high detectionsensitivity and a high amplification speed or to be specificallyamplifiable when amplifying long chain DNA (, which need not bereverse-transcribed and elongated) in the sample, a test similar to thatof the comparative example described above was carried out using each asthe LAMP enzyme, and the presence/absence and specificity of theamplification were evaluated. The results are shown in Table 1.

TABLE 1 Specific Ampli- ampli- Strand displacement enzyme ficationfication Comparative Gsp SSD DNA polymerase + − example Bst DNApolymerase + − Csa DNA polymerase + − 96-7 DNA polymerase − OmniAmp DNApolymerase − Bst2.0 WarmStart DNA polymerase + − Bst3.0 DNA polymerase +− Displace Ace DNA polymerase − SD polymerase − TOPOTAQ DNA polymerase −Embodiments Tin(exo-) DNA polymerase + + Bsm DNA polymerase + +

In the table, the item “Presence/absence of amplification” indicates theresult of turbidity measurement, in which the sign means that the risein turbidity was observed when the concentration of synthetic miRNA iswas 10^5 copies/25 μL, and the sign “−” means that the rise in turbiditywas not observed and therefore the amplification could not achieved. Theitem “specific amplification” indicates the result of electrophoresis,in which the sign “+” means that the specific amplification was possibleunder the condition that the concentration of miRNA is 10^4 copies/25 μLor less, and the sign means that the specific amplification was notpossible under the above-indicated condition. Note that those of thesamples in which the amplification could not be achieved were notsubjected to electrophoresis. The results shown in Table 1 show thatwith Tin(exo-) DMA polymerase or Bsrn DMA polymerase, specificamplification was possible in a reaction liquid containing 1E+4 (10^4)copies of miRNA or less.

It has been found from these test that with Tin(exo-) DNA polymerase orBsrn DNA polymerase, such a method is provided that miRNA, which iscontained in a very small amount in a sample, of a short-chain andeasily decomposable, can be amplified and detected simply with highsensitivity and specificity.

While certain embodiments have been described, these embodiments havebeen presented by way of example only, and are not intended to limit thescope of the inventions. Indeed, the novel embodiments described hereinmay be embodied in a variety of other forms; furthermore, variousomissions, substitutions and changes in the form of the embodimentsdescribed herein may be made without departing from the spirit of theinventions. The accompanying claims and their equivalents are intendedto cover such forms or modifications as would fall within the scope andspirit of the inventions.

What is claimed is:
 1. A method of amplifying a target RNA containing afirst sequence, in a sample, the method comprising: (a)reverse-transcribing the first sequence in the target RNA using areverse-transcription primer which contains a reverse-transcriptionprimer sequence and a first amplification sequence, thereby obtaining areverse-transcription product containing a first' sequence complementaryto the first sequence; (b) dissociating the reverse-transcriptionproduct and the target RNA from each other; (c) hybridizing anelongation primer and the reverse-transcription product to elongate theelongation primer and the reverse-transcription product, therebyobtaining a double-stranded DNA elongation product which contains thefirst' sequence and/or a complementary sequence thereof, wherein theelongation primer contains an elongation primer sequence complementaryto a continuous sequence containing a 3′ terminal region of thereverse-transcription product and a second amplification sequence; and(d) maintaining an amplification reaction liquid containing theelongation product, a LAMP primer set to be bound to the elongationproduct, and Bsm DNA polymerase under a LAMP amplification reactioncondition, thereby amplifying the first' sequence and/or thecomplementary sequence thereof using the elongation product as atemplate.
 2. The method of claim 1, wherein the reverse-transcribingproduct of step (a) is obtained by maintaining a reverse-transcriptionreaction liquid under a reverse-transcription reaction condition, wherethe reverse-transcription reaction liquid includes the sample, a reversetranscriptase, and the reverse-transcription primer.
 3. The method ofclaim 1, wherein the elongation product of Step (c) is obtained bymaintaining an elongation reaction liquid under an elongation reactioncondition, where the elongation reaction liquid includes thereverse-transcription product, DNA polymerase, and the elongation primerto hybridize the elongation primer and the reverse-transcriptionproduct, thereby elongating the elongation primer and thereverse-transcription product using each other as a template.
 4. Themethod of claim 3, wherein the elongation product is a double-strandedDNA, where one of strands of the elongation product contains acomplementary sequence to the second amplification sequence, the first'sequence and the first amplification sequence, and an other strand ofthe elongation product contains the second amplification sequence, acomplementary sequence to the first' sequence and a complementarysequence to the first amplification sequence.
 5. The method of claim 3,wherein the LAMP primer set bound to the elongation product includes atleast, a first primer to be bound to the first amplification sequence ofthe elongation product or a complementary sequence thereof and a secondprimer to be bound to the second amplification sequence of theelongation product or a complementary sequence thereof.
 6. The method ofclaim 1, wherein the target RNA is a short-chain RNA.
 7. The method ofclaim 3, wherein the DNA polymerase is Bsm DNA polymerase.
 8. A methodof detecting a target RNA containing a first sequence, in a sample, themethod comprising: executing the steps (a) to (d) of the RNAamplification method according to claim 1; and (e) detecting theobtained amplification product during or after maintaining the reactionliquid under the amplification reaction condition in step (d).
 9. Anassay kit for amplifying or detecting a target RNA containing a firstsequence, in a sample according to the method of claim 1, the kitcomprising the reverse-transcription primer for reverse-transcribing thefirst sequence; a reverse transcriptase; the elongation primer forobtaining an elongation product; a LAMP primer set to be bound to theelongation product, containing a primer to be bound to the elongationproduct; and Bsm DMA polymerase.
 10. The assay kit of claim 9, whereinthe reverse-transcription primer is for reverse-transcribing the firstsequence to obtain a reverse-transcription product containing the first'sequence complementary to the first sequence; the elongation primer isfor hybridizing with the reverse-transcription product to elongate theelongation primer and the reverse-trsnscription product by using eachother as a template, and obtain an elongation product in which one ofstrands contains a complementary sequence to the second amplificationsequence, the first' sequence and the first amplification sequence, andan other strand contains the second amplification, a complementarysequence to the first' sequence and a complementary sequence to thefirst amplification sequence; and the LAMP primer set to be bound to theelongation product contains a first primer to be bound to the firstamplification sequence of the elongation product or a complementarysequence thereof, and a second primer to be bound to the secondamplification sequence of the elongation product or a complementarysequence thereof.
 11. The assay kit of claim 9, further comprising: DNApolymerase different from Tin(exo-) DNA polymerase and Bsro DNApolymerase.
 12. The assay kit of claim 9, wherein the LAMP primer set tobe bound to the elongation product is an isothermal amplification LAMPprimer set.
 13. The method of claim 1, wherein the first amplificationsequence comprises B1 sequence and B2 sequence, and the secondamplification sequence comprises F1 sequence and F2 sequence, whereinthe B1 sequence, the B2 sequence, the F1 sequence, and the F2 sequence,and B1c sequence, B2c sequence, F1c sequence, and F2c sequence, as theircomplementary sequences respectively, are for the LAMP primer set tobind, the LAMP primer set includes at least a FIP primer and a BIPprimer, the FIP primer comprises the F2 sequence and the F1c sequence,and the BIP primer comprises the B2 sequence and the B1c sequence. 14.The method of claim 13, wherein the first amplification sequence furthercomprises B3 sequence, and the second amplification sequence furthercomprises F3 sequence, wherein the B3 sequence and B3c sequence, and theF3 sequence and F3c sequence, as their complementary sequencesrespectively, are for the LAMP primer set to bind, the LAMP primer setfurther includes a B3 primer and a F3 primer, the B3 primer comprisesthe B3 sequence, and the F3 primer comprises the F3 sequence.
 15. Themethod of claim 8, wherein the reverse-transcribing product of step (a)is obtained by maintaining a reverse-transcription reaction liquid undera reverse-transcription reaction condition, where thereverse-transcription reaction liquid includes the sample, a reversetranscriptase, and the reverse-transcription primer.
 16. The method ofclaim 8, wherein the elongation product of Step (c) is obtained bymaintaining an elongation reaction liquid under an elongation reactioncondition, where the elongation reaction liquid includes thereverse-transcription product, DNA polymerase, and the elongation primerto hybridize the elongation primer and the reverse-transcriptionproduct, thereby elongating the elongation primer and thereverse-transcription product using each other as a template.
 17. Themethod of claim 16, wherein the elongation product is a double-strandedDNA, where one of strands of the elongation product contains acomplementary sequence to the second amplification sequence, the first'sequence and the first amplification sequence, and an other strand ofthe elongation product contains the second amplification sequence, acomplementary sequence to the first'0 sequence and a complementarysequence to the first amplification sequence.
 13. The method of claim16, wherein the LAMP primer set bound to the elongation product includesat least a first primer to be bound to the first amplification sequenceof the elongation product or a complementary sequence thereof and asecond primer to be bound to the second amplification sequence of theelongation product or a complementary sequence thereof.
 19. The methodof claim 8, wherein the target RNA is a short-chain RNA.
 20. The methodof claim 16, wherein the DNA polymerase is Bsm DMA polymerase.
 21. Themethod of claim 8, wherein the first amplification sequence comprises B1sequence and B2 sequence, and the second amplification sequencecomprises F1 sequence and F2 sequence, wherein the B1 sequence, the B2sequence, the F1 sequence and the F2 sequence, and B1c sequence, B2csequence, F1c sequence and F2c sequence, as their complementarysequences respectively, are for the LAMP primer set to bind, the LAMPprimer set includes at least a FIP primer and a BIP primer, the FIPprimer comprises the F2 sequence and the F1c sequence, and the BIPprimer comprises the B2 sequence and the B1c sequence.
 22. The method ofclaim 21, wherein the first amplification sequence further comprises B3sequence, and the second amplification sequence further comprises F3sequence, wherein the B3 sequence and B3c sequence, and the F3 sequenceand F3c sequence, as their complementary sequences respectively, are forthe LAMP primer set to bind, the LAMP primer set further includes a B3primer and a F3 primer, the B3 primer comprises the B3 sequence, and theF3 primer comprises the F3 sequence.
 23. The kit of claim 9, wherein theLAMP primer set includes at least a FIP primer and a BIP primer, the FIPprimer comprises the F2 sequence and the F1c sequence, and the BIPprimer comprises the B2 sequence and the B1c sequence.
 24. The kit ofclaim 23, wherein the LAMP primer set further includes a B3 primer and aF3 primer, the B3 primer comprises the B3 sequence, and the F3 primercomprises the F3 sequence.